Nonintercalating Fluors for Nucleic Acid Probes

This Phase l is a feasibility study of the design and synthesis of novel, non-intercalative dyes for nucleic acid (DNA/RNA) probes for DNA hybridization and other nucleic acid quantitation assays. In the past, radiolabels were used for detecting hybrids, but these are being replaced by non-radioactive probes such as oligonucleotide probes linked to fluorescent dyes. Fluorescent probes have been partially successful, but there remain significant problems associated with accurate fluorescent quantitation. Most fluorescent indicators are planar molecules that can intercalate between similarly flat base-pairs of nucleic acid helices, resulting in the quenching of fluorescence with consequent errors in quantitation, as well as loss in sensitivity of detection. We will develop new types of fluorescent dyes that are sterically hindered so as not to intercalate into the DNA helices when they are linked to probe sequences. Three approaches will be taken: 1. a sterically hindered fluorophore will be developed, 2. a "dendritic" spacer unit will be developed to prevent intercalation of linked dyes and to allow multiple dye attachment, thus enabling signal multiplication, and 3. a fluorophore will be entrapped within a caged system, or host-guest complex. The new fluorophores will be attached to deoxyuridine (dU) or deoxyuridine triphosphate (dUTP), which will be incorporated into oligonucleotide probes, the efficacy of which can be easily determined.